Unit 1 Lecture-Genes

8/10/2019 Unit 1 Lecture-Genes

1/36

Transcription and Translation

The Relationship

Between Genes and

Proteins

8/10/2019 Unit 1 Lecture-Genes

2/36

Table of Contents

History: linking genes and proteins

Getting from gene to protein: transcription Evidence for mRNA

Overview of transcription

RNA polymerase

Stages of Transcription Promoter recognition

Chain initiation

Chain elongation

Chain termination

mRNA Synthesis/Processing References

8/10/2019 Unit 1 Lecture-Genes

3/36

Table of Contents (continued)

Getting from gene to protein: genetic code

Getting from gene to protein: translation Translation Initiation

Translation Elongation

Translation Termination

References

8/10/2019 Unit 1 Lecture-Genes

4/36

History: linking genes and proteins

1900s Archibald Garrod

Inborn errors of metabolism: inherited human metabolic diseases(more information)

Genes are the inherited factors

Enzymes are the biological molecules that drive

metabolic reactions

Enzymes are proteins

Question:

How do the inherited factors, the genes, control the structure and

activity of enzymes (proteins)?

8/10/2019 Unit 1 Lecture-Genes

5/36

History: linking genes and proteins

Beadle and Tatum (1941) PNAS USA 27, 499506.

Hypothesis: If genes control structure and activity of metabolic enzymes, then

mutations in genes should disrupt production of required nutrients,

and that disruption should be heritable.

Method:

Isolated ~2,000 strains from single irradiate spores (Neurospora)

that grew on rich but not minimal medium. Examples: defects in B1,

B6 synthesis.

Conclusion:

Genes govern the ability to synthesize amino acids, purines andvitamins.

8/10/2019 Unit 1 Lecture-Genes

6/36

History: linking genes and proteins

1950s: sickle-cell anemia

Glu to Val change in hemoglobin Sequence of nucleotides in gene determines sequence of amino

acids in protein

Single amino acid change can alter the function of the protein

Tryptophan synthase gene in E. coli

Mutations resulted in single amino acid change

Order of mutations in gene same as order of affected amino acids

8/10/2019 Unit 1 Lecture-Genes

7/36

From gene to protein: transcription

Gene sequence (DNA) recopied or transcribed to RNA

sequence Product of transcription is a messenger molecule that

delivers the genetic instructions to the protein synthesis

machinery: messenger RNA (mRNA)

8/10/2019 Unit 1 Lecture-Genes

8/36

Transcription: evidence for mRNA

Brenner, S., Jacob, F. and Meselson, M. (1961) Nature

190, 57681. Question: How do genes work?

Does each one encode a different type of ribosome which in turn

synthesizes a different protein, OR

Are all ribosomes alike, receiving the genetic information to create

each different protein via some kind of messenger molecule?

8/10/2019 Unit 1 Lecture-Genes

9/36

Transcription: evidence for mRNA

E. colicells switch from making bacterial proteins to

phage proteins when infected with bacteriophage T4. Grow bacteria on medium containing heavy nitrogen

(15N) and carbon (13C).

Infect with phage T4.

Immediately transfer to light medium containingradioactive uracil.

8/10/2019 Unit 1 Lecture-Genes

10/36

Transcription: evidence for mRNA

If genes encode different ribosomes, the newly

synthesized phage ribosomes will be light. If genes direct new RNA synthesis, the RNA will contain

radiolabeled uracil.

Results:

Ribosomes from phage-infected cells were heavy, banding at thesame density on a CsCl gradient as the original ribosomes.

Newly synthesized RNA was associated with the heavy ribosomes.

New RNA hybridized with viral ssDNA, not bacterial ssDNA.

8/10/2019 Unit 1 Lecture-Genes

11/36

Transcription: evidence for mRNA

Conclusion

Expression of phage DNA results in new phage-specific RNAmolecules (mRNA)

These mRNA molecules are temporarily associated with ribosomes

Ribosomes do not themselves contain the genetic directions for

assembling individual proteins

8/10/2019 Unit 1 Lecture-Genes

12/36

Transcription: overview

Transcription requires:

ribonucleoside 5 triphosphates:ATP, GTP, CTP and UTP

bases are adenine, guanine, cytosine and uracil

sugar is ribose (not deoxyribose)

DNA-dependent RNA polymerase Template (sense) DNA strand

Animation of transcription

8/10/2019 Unit 1 Lecture-Genes

13/36

Transcription: overview

Features of transcription:

RNA polymerasecatalyzes sugar-phosphate bondbetween 3-OH of ribose and the 5-PO4.

Order of bases in DNA template strand determines order

of bases in transcript.

Nucleotides are added to the 3-OH of the growing chain. RNA synthesis does not require a primer.

8/10/2019 Unit 1 Lecture-Genes

14/36

Transcription: overview

In prokaryotes transcription and translation are coupled.

Proteins are synthesized directly from the primarytranscript as it is made.

In eukaryotes transcription and translation are separated.

Transcription occurs in the nucleus, and translation occurs

in the cytoplasm on ribosomes. Figurecomparing eukaryotic and prokaryotic transcription

and translation.

8/10/2019 Unit 1 Lecture-Genes

15/36

Transcription: RNA Polymerase

DNA-dependent

DNA template, ribonucleoside 5 triphosphates, and Mg2+

Synthesizes RNA in 5 to 3 direction

E. coliRNA polymerase consists of 5 subunits

Eukaryotes have three RNA polymerases

RNA polymerase II is responsible for transcription of protein-codinggenes and some snRNA molecules

RNA polymerase II has 12 subunits

Requires accessory proteins (transcription factors)

Does not require a primer

8/10/2019 Unit 1 Lecture-Genes

16/36

Stages of Transcription

Promoter Recognition

Chain Initiation Chain Elongation

Chain Termination

8/10/2019 Unit 1 Lecture-Genes

17/36

Transcription: promoter recognition

Transcription factors bind to promoter sequences and

recruit RNA polymerase. DNA is bound first in a closed complex. Then, RNA

polymerase denatures a 1215 bp segment of the DNA

(open complex).

The site where the first base is incorporated into thetranscription is numbered +1 and is called the

transcription start site.

Transcription factors that are required at every promoter

site for RNA polymerase interaction are called basaltranscription factors.

8/10/2019 Unit 1 Lecture-Genes

18/36

Promoter recognition: promoter sequences

Promoter sequences vary considerably.

RNA polymerase binds to different promoters withdifferent strengths; binding strength relates to the level of

gene expression

There are some common consensus sequencesfor

promoters: Example: E. coli35 sequence (found 35 bases 5 to the start of

transcription)

Example: E. coliTATA box (found 10 bases 5 to the start of

transcription)

8/10/2019 Unit 1 Lecture-Genes

19/36

Promoter recognition: enhancers

Eukaryotic genes may also have enhancers.

Enhancers can be locatedat great distances from thegene they regulate, either 5 or 3 of the transcription

start, in introns or even on the noncoding strand.

One of the most common ways to identify promoters and

enhancers is to use a reporter gene.

8/10/2019 Unit 1 Lecture-Genes

20/36

Promoter recognition: other players

Many proteins can regulate gene expression by

modulating the strength of interaction between thepromoter and RNA polymerase.

Some proteins can activate transcription (upregulate gene

expression).

Some proteins can inhibit transcription by blockingpolymerase activity.

Some proteins can act both as repressors and activators

of transcription.

8/10/2019 Unit 1 Lecture-Genes

21/36

Transcription: chain initiation

Chain initiation:

RNA polymerase locally denatures the DNA. The first base of the new RNA strand is placed

complementary to the +1 site.

RNA polymerase does not require a primer.

The first 8 or 9 bases of the transcript are linked.Transcription factors are released, and the polymerase

leaves the promoter region.

Figure of bacterial transcription initiation.

8/10/2019 Unit 1 Lecture-Genes

22/36

Transcription: chain elongation

Chainelongation:

RNA polymerase moves along the transcribed or templateDNA strand.

The new RNA molecule (primary transcript) forms a short

RNA-DNA hybrid molecule with the DNA template.

8/10/2019 Unit 1 Lecture-Genes

23/36

Transcription: chain termination

Most known about bacterial chain termination

Termination is signaled by a sequence that can form ahairpin loop.

The polymerase and the new RNA molecule are released

upon formation of the loop.

Review the transcription animation.

8/10/2019 Unit 1 Lecture-Genes

24/36

Transcription: mRNA synthesis/processing

Prokaryotes: mRNA transcribed directly from DNA

template and used immediately in protein synthesis Eukaryotes: primary transcript must be processedto

produce the mRNA

Noncoding sequences (introns) are removed

Coding sequences (exons) spliced together

5-methylguanosine cap added

3-polyadenosine tail added

8/10/2019 Unit 1 Lecture-Genes

25/36

Transcription: mRNA synthesis/processing

Removal of introns and splicing of exons can occur

several ways For introns within a nuclear transcript, a spliceosomeis required.

Splicesomes protein and small nuclear RNA (snRNA)

Specificity of splicing comes from the snRNA, some of which contain

sequences complementary to the splice junctions between introns and

exonsAlternative splicingcan produce different forms of a protein from

the same gene

Mutationsat the splice sites can cause disease

Thalassemia Breast cancer(BRCA 1)

8/10/2019 Unit 1 Lecture-Genes

26/36

Transcription: mRNA synthesis/processing

RNA splicing inside the nucleus on particles called

spliceosomes. Splicesomes are composed of proteins and small RNA

molecules (100200 bp; snRNA).

Both proteins and RNA are required, but some suggesting

that RNA can catalyze the splicing reaction. Self-splicing in Tetrahymena: the RNA catalyzes its own

splicing

Catalytic RNA: ribozymes

8/10/2019 Unit 1 Lecture-Genes

27/36

From gene to protein: genetic code

Central Dogma

Information travels from DNA to RNA to Protein Is there a one-to-one correspondence between DNA, RNA and Protein?

DNA and RNA each have four nucleotides that can form them; so yes, there

is a one-to-one correspondence between DNA and RNA.

Proteins can be composed of a potential 20 amino acids; only four RNA

nucleotides: no one-to-one correspondence.

How then does RNA direct the order and number of amino acids in a protein?

8/10/2019 Unit 1 Lecture-Genes

28/36

From gene to protein: genetic code

How many bases are required for each amino acid?

(4 bases)

2bases/aa

= 16 amino acidsnot enough (4 bases)3bases/aa= 64 amino acid possibilities

Minimum of 3 bases/aa required

What is the nature of the code?

Does it have punctuation? Is it overlapping?

Crick, F.H. et al. (1961) Nature192, 122732.

(http://profiles.nlm.nih.gov/SC/B/C/B/J/)

3-base, nonoverlapping code that is read from a fixed point.

8/10/2019 Unit 1 Lecture-Genes

29/36

From gene to protein: genetic code

Nirenberg and Matthaei: in vitro protein translation

Found that adding rRNA prolonged cell-free protein synthesisAdding artificial RNA synthesized by polynucleotide phosphorylase

(no template, UUUUUUUUU) stimulated protein synthesis more

The protein that came out of this reaction was polyphenylalanine

(UUU = Phe)

Other artificial RNAs: AAA = Lys; CCC =Pro

8/10/2019 Unit 1 Lecture-Genes

30/36

From gene to protein: genetic code

Nirenberg:

Triplet binding assay: add triplet RNA, ribosomes, binding factors,GTP, and radiolabeled charged tRNA (figure)

UUU trinucleotide binds to Phe-tRNA

UGU trinucleotide binds to CYS-tRNA

By fits and starts the triplet genetic codewas worked out.

Each three-letter word (codon) specifies an amino acid ordirections to stop translation.

The code is redundant or degenerate: more than one way to

encode an amino acid

8/10/2019 Unit 1 Lecture-Genes

31/36

From gene to protein: Translation

Components required for translation:

mRNA Ribosomes

tRNA

Aminoacyl tRNA synthetases

Initiation, elongation and termination factors

Animation of translation

8/10/2019 Unit 1 Lecture-Genes

32/36

Translation: initiation

Ribosome small subunit binds to mRNA

Charged tRNA anticodon forms base pairs with the mRNAcodon

Small subunit interacts with initiation factors and special

initiator tRNA that is charged with methionine

mRNA-small subunit-tRNA complex recruits the largesubunit

Eukaryoticand prokaryoticinitiation differ slightly

8/10/2019 Unit 1 Lecture-Genes

33/36

Translation: initiation

The large subunit of the ribosome contains three binding

sitesAmino acyl (A site)

Peptidyl (P site)

Exit (E site)

At initiation,

The tRNAfMetoccupies the P site

A second, charged tRNA complementary to the next codon binds

the A site.

8/10/2019 Unit 1 Lecture-Genes

34/36

Translation: elongation

Elongation

Ribosome translocates by three bases after peptide bondformed

New charged tRNA aligns in the A site

Peptide bondbetween amino acids in A and P sites is

formed Ribosome translocates by three more bases

The uncharged tRNA in the A site is moved to the E site.

8/10/2019 Unit 1 Lecture-Genes

35/36

Translation: elongation

EF-Tu recruits charged tRNA to A site. Requires

hydrolysis of GTP Peptidyl transferase catalyzes peptide bond formation

(bond between aa and tRNA in the P site converted to

peptide bond between the two amino acids)

Peptide bond formation requires RNA and may be aribozyme-catalyzed reaction

8/10/2019 Unit 1 Lecture-Genes

36/36

Translation: termination

Termination

Elongation proceeds until STOP codon reached UAA, UAG, UGA

No tRNA normally exists that can form base pairing with a

STOP codon; recognized by a release factor

tRNA charged with last amino acid will remain at P site Release factors cleave the amino acid from the tRNA

Ribosome subunits dissociate from each other

Review the animation of translation